Method of producing and purifying soluble recombinant coq5 protein and soluble recombinant coq5 protein thereof

ABSTRACT

The present invention relates to a producing and purifying method of soluble recombinant COQ5 protein, which is expressed in soluble form by  E. coli , under native conditions. The method is characterized by pre-treating bacterial lysate with low concentration of ionic detergent, such as SDS, before purification; and the purifying method is performed under native condition without using urea to avoid the problems of requiring lengthy processes to remove urea in purified protein solution or re-aggregation and precipitation of protein after removal of urea.

RELATED APPLICATIONS

This application is a Divisional patent application of co-pendingapplication Ser. No. 13/600,963, filed on 31 Aug. 2012, now pending. Theentire disclosure of the prior application Ser. No. 13/600,963, fromwhich an oath or declaration is supplied, is considered a part of thedisclosure of the accompanying Divisional application and is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a producing and purifying method ofrecombinant COQ5 protein expressed in soluble form by E. coli. Thepurifying method is characterized by pre-treating bacterial lysate withlow concentration of ionic detergent before purification; and performedin non-denaturing conditions to avoid the problems of urea removal frompurified protein solution if purified under denaturing condition and ofre-aggregation or precipitation of the purified protein upon removal ofurea or during storage.

BACKGROUND OF THE INVENTION

Many human recombinant proteins form inclusion body or non-native formwhen expressed in E. coli and so denaturing conditions, such as the useof 8 M urea, have to be used to purify protein, which may cause troubleduring protein purification and preparation of purified protein beforeantibody production than protein purified as soluble protein or undernative conditions. For example, the procedures to purify proteins ininclusion body are more complicated than that for purifying solubleproteins. Moreover, if denaturing conditions are used to purifyproteins, removal of urea in purified protein before antibody productionis very troublesome, such as the use of lengthy buffer-exchangeprocesses with concentrators, or even SDS-PAGE purification.Furthermore, very often proteins purified under denaturing conditionsmay become aggregated or form precipitation once the denaturing agent isremoved and the protein is concentrated, which cause problems byreducing amount of protein yield available for antibody production andsubsequent affinity purification of antibody. Attempts to refold suchpurified protein back to native form often do not work well or aredifficult. On the other hand, only soluble recombinant protein, but notaggregated protein, can be used for pharmaceutical purposes and to studythe biochemical properties, biological functions, and crystal structure.

Although antibodies generated against non-native form of protein orsynthetic peptide for specific epitopes can be used for the purpose ofWestern blot analysis, the use of native protein as the antigen mightproduce more useful polyclonal antibody against dominant epitopespresented by natively folded protein. Moreover, antibody that canrecognize epitopes the folded structure of mature protein would beneeded for immunoprecipitation assay. Multiple try-and-error may beneeded when only aiming at specific epitopes by synthetic peptides.Moreover, denatured recombinant proteins are often in aggregated orprecipitated form, which cause low yield and high cost if using solublefraction to immunize rabbits and is not suitable for furtherpurification of antibody by affinity column.

Although administration of exogenous coenzyme Q₁₀ has been extensivelyused, the biosynthesis for endogenous coenzyme Q₁₀ and mechanisms forcoenzyme Q₁₀ deficiency diseases in humans are poorly studied. The lackof validated commercial antibodies and soluble purified recombinantproteins for human PDSS and COQ proteins essential for terminalbiosynthetic reactions of coenzyme Q₁₀ has been an obstacle in thisarea. COQ5 protein has been found to be essential as one of the nine COQproteins essential for coenzyme Q₆ biosynthesis in yeast, but its rolein humans for biosynthesis of coenzyme Q₁₀ has not been studied. YeastCOQ5 protein is a methyltransferase and a mitochondrial matrix proteinassociated with inner membrane that can form a multi-subunit complexwith other COQ proteins in yeast (Baba et al., J Biol Chem 279: 10052,2004). However, there has been no study on human COQ5 protein.

For making polyclonal antibody against nuclear DNA-encoded mitochondrialmatrix protein by using recombinant protein as the antigen, most peoplejust expressed full-length protein in E. coli. However, that shouldresult in misfolded protein and formation of inclusion body becauseremoval of mitochondrial targeting signal (MTS) by peptidase inmitochondria after import of mitochondrial proteins into matrix isnecessary for correct folding of the protein into mature structure andbacteria do not have such machinery (Chacinska et al., Cell 138: 628,2009). We have previously tried to purify His-tagged full-length humanCOQ5 protein expressed in E. coli, but it formed inclusion under allconditions commonly employed. We could only purify that recombinantprotein under denaturing condition with 8 M urea, but proteinprecipitated severely once urea was removed by buffer exchange withphosphate-buffer saline (PBS). Nevertheless, one publication hascompared and indicated that removal of N-terminal MTS of mouseendonuclease G, a mitochondrial matrix protein, could increase thesolubility of the protein when its mutant was expressed in E. colialthough no data were shown for the comparison (Yoon et al., Acta.Crystallogr. Sect. F. Struct Biol. Cryst Commun 65: 504, 2009).

So far, other than Baba's article on yeast COQ5 (Baba et al., J BiolChem 279: 10052, 2004), no publication has reported the purification ofCOQ5 protein for generation of antibody. Although they indeed expressedmature form of recombinant yeast COQ5 protein without N-terminal MTS inE. coli, denaturing conditions with the use of urea followed by SDS-PAGEpurification were still applied for purification of recombinant yeastCOQ5 proteins. The antibody produced by this group against suchdenatured COQ5 protein was not affinity-purified either (Baba et al., JBiol Chem 279: 10052, 2004). These results indicated that additionalstrategy might be needed to purify yeast COQ5 protein as solubleprotein. Because yeast COQ5 protein is not a soluble matrix protein butis associated with other protein on mitochondrial inner membrane as aninsoluble complex (Baba et al., J Biol Chem 279: 10052, 2004), wesuspected that even if MTS-truncated could be expressed as native formin the cytoplasm of E. coli, it might become associated with plasmamembrane of bacteria to form insoluble complex. The rationale is thatmitochondria in modern eukaryotes is from ancient bacteria duringevolution and so biochemical characteristics of mitochondrial proteinsin mitochondrial matrix and inner membrane are often similar to that incytoplasm and plasma membrane of bacteria, respectively (Pallen, TrendsMicrobiol 19: 58, 2011).

The earliest commercial antibodies for human COQ5 available were fromAbgent (Cat.#: AP9319c) and ProteinTech (Cat.#: 17453-1-AP), althoughmore commercial antibodies came out in 2012 with similar description asthat in datasheets of these two antibodies. By using information fromonline datasheets of these two antibodies from Abgent and ProteinTech asexamples, we could find many problems for those commercial antibodies.For example, they did not prove the detection of endogenous human COQ5protein by these antibodies, but just used mouse tissues asdemonstration. No validation experiments, such as overexpression orknockdown of COQ5 gene in cells, were performed to confirm thespecificity either. Moreover, the size of protein detected by Westernblot by antibodies from different sources was not the same. On the otherhand, the antigen of ProteinTech's antibody, a His-tagged full-lengthhuman COQ5 protein containing MTS, is also a commercial product (Cat. #:ag10203). Although no description about the purification condition couldbe found on the datasheet of the antigen, it appears that this purifiedrecombinant protein is not soluble in aqueous solution without ionicdetergent as the protein is resolved in PBS containing the detergentSarcosyl (N-laurylsarcosine) at the concentration of 0.7%.

SUMMARY OF THE INVENTION

This invention is based on the discovery that treating bacteria celllysate with low concentration of an ionic detergent, such as SDS, mightassist the dissociation of MTS-truncated human COQ5 protein frombacteria membrane in addition to lysing bacteria with lysozyme and DNaseI. By doing this way, the purified MTS-truncated human COQ5 proteincould remain in soluble form without any aggregation and precipitationat 4° C. for several days or after being stored at −20° C. for severalmonths.

Thus, in one aspect, the present invention provides a producing andpurifying method of soluble recombinant COQ5 protein under nativeconditions. The method of the invention comprises steps of: providing anE. coli host cell transformed with an expression vector containing DNAfragments that encode sequence for soluble COQ5 protein; inducing theexpressing of recombinant soluble COQ5 protein in the E. coli host cell;lysing the bacterial cells by a lysis buffer comprising lysozyme andDNase I; treating the cell lysate with low concentration of ionicdetergent; centrifugation for removing insoluble proteins or debris andcollecting supernatant containing the recombinant soluble COQ5 protein;and applying the supernatant to Ni-NTA beads under native condition(without using urea or any other detergents) to purify the recombinantsoluble COQ5 protein.

In certain embodiments of this invention, the said soluble COQ5 proteinis a mitochondrial targeting signal (MTS)-truncated human COQ5 protein.In other embodiments of this invention, the said soluble COQ5 protein isa MTS-truncated human COQ5 protein with His tag at N-terminus,comprising the amino acid sequence of SEQ ID NO. 2.

In certain embodiments of this invention, the said E. coli host cell isinduced to express recombinant protein at a temperature in the range of10-24° C. In other embodiments of this invention, the said E. coli hostcell is induced to express recombinant protein at 16° C.

In certain embodiments of this invention, the low concentration of ionicdetergent comprises no more than 0.3% of sodium dodecyl sulfate (SDS).In other embodiments of this invention, the final concentration of SDSin the lysate solution is 0.05% to 0.1%.

In another aspect of the invention, it relates to a recombinant solubleCOQ5 protein produced by the method of this invention. In certainembodiments of this invention, the said soluble COQ5 protein is aMTS-truncated human COQ5 protein. The recombinant soluble COQ5 proteinobtained according to the producing and purifying method described abovedoes not become aggregated or form precipitation once solution duringpurification is exchanged with PBS buffer and the protein isconcentrated, which may improve protein yield available for antibodyproduction and subsequent affinity purification of anti-COQ5 antibodies.

Other features or advantages of the present invention will be apparentfrom the following drawings and detailed description of severalembodiments, and also from the appending claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the recovery of recombinant full-length human COQ5 protein(His-hCOQ5) during purification under native condition MTS-truncatedhuman COQ5 protein determined by SDS-PAGE (A) and Western blot analysiswith anti-His antibody (B). The predicted size of His-hCOQ5 fusionprotein is 39.4 kD. Protein on Ni-NTA beads were washed (WL, WI, and W2)and eluted (E1 and E2) by 20 mM Tris-Cl buffer (pH 8.0) containingdifferent concentrations of imidazole. P: pellet collected aftercentrifugation of bacterial lysate, WL: 10 mM imidazole, W1: 20 mMimidazole, W2: 50 mM imidazole, E1: 100 mM imidazole, E2: 300 mMimidazole. Results indicate that His-hCOQ5 formed inclusion body whenpurified under native condition as it could only found in pellet frombacterial lysate.

FIG. 2 shows the recovery of recombinant MTS-truncated human COQ5protein (His-ΔMTS-hCOQ5) during the purification process under nativecondition determined by SDS-PAGE (A) and Western blot analysis usinganti-His antibody (B). The predicted size of His-ΔMTS-hCOQ5 fusionprotein is 34 kD. Protein on Ni-NTA beads were washed (WL, WI, and W2)and eluted (E1 and E2) by 20 mM Tris-Cl buffer (pH 8.0) containingdifferent concentrations of imidazole. P: pellet collected aftercentrifugation of bacterial lysate, WL: 10 mM imidazole, W1: 20 mMimidazole, W2: 50 mM imidazole, E1: 100 mM imidazole, E2: 300 mMimidazole. The His-ΔMTS-hCOQ5 protein was mostly present in the fractionfrom two eluting solutions from supernatant of bacterial lysate.

FIG. 3 demonstrates formation of aggregation and precipitation inHis-hCOQ5 protein solution, but not His-ΔMTS-hCOQ5 protein solutionafter buffer exchange of protein solutions with PBS by a centrifugalconcentrator. (A) Results of wavelength scan showing the absorbance(y-axis) to wavelength (x-axis) relationship of two protein solutionsafter 10-fold dilution with PBS were used to demonstrate the presence ofaggregation in His-hCOQ5 solution after forth buffer exchange with PBS.Dashed line and solid line indicated the spectrums for His-hCOQ5 andHis-ΔMTS-hCOQ5, respectively. (B) High degree of protein precipitationafter fifth buffer exchange could be easily visualized. Left and rightphotographs showed the appearance of two protein solutions inmicrocentrifuge tubes before and after centrifugation at 12000×g at 4°C. for 5 min, respectively.

FIG. 4 shows the detection of human COQ5 protein in total lysate,cytosol, and mitochondria of 143B cells by Western blot with theanti-COQ5 antibody. COX II, mtDNA-encoded protein, and α-tubulin,cytosolic cytoskeleton protein, are markers to ensure the lack ofcytosolic protein in mitochondria and the absence of mitochondrialprotein in the cytosolic fraction, respectively.

FIG. 5 shows the verification on the specificity of anti-COQ5 antibodyon recognizing full-length and mature form of human COQ5 protein in 143Bcells by Western blot following transient overexpression (A) andknockdown of COQ5 gene (B). Detection of α-tubulin was performed as aloading control. The predicted molecular weight of full-length COQ5protein (FL-COQ5) and mature COQ5 without MTS (ΔMTS-COQ5) was 37.1 and31.8 kD, respectively.

DETAILED DESCRIPTION OF THE INVENTION

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications cited herein arehereby incorporated by reference in their entirety. Further, anymechanism proposed below does not in any way restrict the scope of theclaimed invention.

Example Example 1 Production and Purification of Soluble RecombinantHuman COQ5 Protein Construction of Vector for Expressing MTS-TruncatedHuman COQ5 Protein

Accession number for reference sequence human COQ5 gene and COQ5 proteinis NM_(—)032314 and NP_(—)115690, respectively, from GenBank in NationalCenter for Biotechnology Information (NCBI). Because the exact length ofMTS for human COQ5 protein has not been reported, the length ofN-terminal MTS for human COQ5 protein was predicted by using thereference protein sequence of COQ5 protein and three online tools:Target P (http://www.cbs.dtu.dk/services/TargetP/), MitoProt II(http://ihg.gsf.de/ihg/mitoprot.html) and UniprotKB(http://www.uniprot.org/uniprot/). However, due to different algorithmsused by different programs, the results of prediction from three onlinetools are often different and may not match with real MTS occurred incells. Predicted lengths of MTS for human COQ5 protein were 17, 42, and49 amino acids in length from three different online tools. The longestlength of MTS predicted by UniprotKB was chosen for the construction ofexpression plasmid.

DNA segment of the open reading frame (ORF) of human COQ5 gene, +148 to+984 nt, without the first 147 nt encoding MTS was generated by PCR byusing cDNA from human 143B cell line as the template with the forwardprimer NdeI-hCOQ5: 5′-GGGAATTCCATATGGCGGAAACGCACTTTGGG-3′; SEQ ID NO: 3;and reverse primer hCOQ5-XhoI-R:(5′-GACCGCTCGAGTTAAAGTTTGAAGCCAGAATG-3′); SEQ ID NO: 4. The NdeI-hCOQ5primer contained an NdeI restriction site (underlined sequence) followedby +148 to +165 of COQ5 ORF. The hCOQ5-XhoI-R primer contained an XhoIrestriction site (underlined sequence) followed by +984 to +964 of COQ5ORF containing the stop codon. The size of amplicon from these twoprimers was 862 bp. PCR product was digested with NdeI and XhoI andligated into pET-15b vector (Novagen) to generate pET15b-ΔMTS-hCOQplasmid to express recombinant MTS-truncated human COQ5 protein withN-terminal His-tag (His-ΔMTS-hCOQ5) in E. coli (BL21) DE3. The DNAsequence of the cloned insert was confirmed to be the same as that ofthe reference sequence by direct sequencing. The total length of therecombinant protein expressed by the pET15b-ΔMTS-hCOQ5 plasmid was 299amino acids because it contained not only the 278 amino acids (withoutthe MTS from the full-length protein in 327 amino acids), but also other21 amino acids on the pET15b vector for initiation of translation (Met),His-tag, thrombin cleavage site, NdeI cloning site, and other connectingamino acids on the N-terminus. The molecular weight of this recombinantprotein was predicted to be 34 kD based on the results from the analysison ProtParam (http://web.expasy.org/protparam/).

Expression and Purification of MTS-Truncated Human COQ5 Protein

Expression of His-ΔMTS-hCOQ5 protein in E. coli (BL21) DE3 was inducedby 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16° C. for 8-16hour. Bacterial culture in 2 L were centrifuged at 6000×g at 4° C. andthen bacteria pellet was lysed by lysis buffer containing 20 mM Tris-Clbuffer (pH 8.0) containing 150 mM NaCl, 0.5 mg/mL lysozyme, and 5 μg/mLDNase I at 4° C. for 1 hour. Then SDS stock solution was added into thelysis buffer to a final concentration of 0.1%, and then protein wasincubated at 4° C. for another 8 hours on shaker. To remove insolubleproteins or cell debris, the solution containing protein extract wascentrifuged at 10,000×g at 4° C.

Next, soluble His-ΔMTS-hCOQ5 protein in supernatant was purified bynickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) under nativecondition modified from the protocols in Qiagen's online handbook,QIAexpressionist™ (fifth edition) downloaded fromhttp://www.qiagen.com/HB/QIAexpressionist. In brief, supernatant fromlysate was incubated with Ni-NTA and put into a column followed bypassing through a gradient of imidazole: 10 mM, 20 mM, 50 mM, 100 mM,and 300 mM in 20 mM Tris-Cl buffer (pH 8.0) containing 150 mM NaCl andprotease inhibitor cocktail (BioShop). SDS-PAGE staining with Coomassiebrilliant blue was performed to check purity and quantity of purifiedproteins in each fraction. The presence of His-tag protein was confirmedby Western blot analysis using mouse anti-His antibody (GE) as theprimary antibody and horseradish peroxidase-conjugated goat anti-mouseIgG as the secondary antibody. The purified protein was also confirmedto be human COQ5 protein by the analysis on matrix-assisted laserdesorption/ionization-time of flight (MALDI-TOF) mass spectrometry.

On the other hand, for the comparison, the same vector containingfull-length OFR of human COQ5 for expression of His-tagged full-lengthHis-hCOQ5 (pET15b-hCOQ5) was constructed and the recombinant protein waspurified by Ni-NTA beads under denaturing condition because it was stillpresent as inclusion body when the same lysis conditions andpurification steps under native condition were applied. To obtainpurified His-hCOQ5 under denaturing condition, bacterial pellet waslysed by lysis buffer, which consisted of 20 mM Tris-Cl buffer (pH 8.0),150 mM NaCl, and 8M urea, at 4° C. for 12 hr followed by the samepurification procedures as for native condition except the addition of 8M urea in all washing and elution solutions.

FIGS. 1 and 2 show the purification of recombinant full-length humanCOQ5 protein (His-hCOQ5) and recombinant ΔMTS-human COQ5 protein(His-ΔMTS-hCOQ5) under native condition determined by SDS-PAGE andWestern blot analysis, respectively. As shown in FIG. 1, recombinantfull-length human COQ5 protein could not be purified under nativeconditions, even thought bacteria were grown at 16° C. for proteininduction and its lysate was pretreated with lysis buffer containing0.1% SDS. However, recombinant MTS-truncated human COQ5 protein could bepurified under native conditions when expression was induced at 16□ andwhen bacteria were treated by lysis buffer containing 0.1% SDS beforepurification to (FIG. 2).

Example 2 Production of Rabbit Polyclonal Antibodies Against Human COQ5(hCOQ5) Protein by Using the Purified Recombinant ΔMTS-hCOQ5 ProteinConcentration and Removal of Imidazole in Purified His-ΔMTS-hCOQ5Protein by a Centrifugal Concentrator

Before antibody production, purified His-ΔMTS-hCOQ5 protein was furtherprocessed by using Vivaspin 6 Ultrafiltration Device with 3000 MWCO (GE)to concentrate protein and to remove imidazole. Fractions from elutionof Ni-NTA beads containing purified His-ΔMTS-hCOQ5 protein were pooledand concentrated to 2 mL by centrifugation at 3000×g at 4° C. for 10minutes. The concentrated protein solution (2 ml) in concentrator bodywas mixed with 2 mL of phosphate-buffered saline (PBS) containingprotease inhibitor (PI) to reduce concentration of imidazole and thenwas centrifuged again to the volume of 2-3 ml solution in concentratorbody. This buffer exchange step was repeated for another four times. Thefinal protein solution was then sent to GeneTex Corporation (Taiwan) togenerate rabbit polyclonal antibody and the subsequent affinitypurification of antibody. The amount of purified recombinantHis-ΔMTS-hCOQ5 protein needed for immunization of rabbits and affinitypurification was 2 mg and 1 mg, respectively. His-ΔMTS-hCOQ5 remained inclear and soluble from without obvious change in protein concentrationfor several days at 4° C. or after thawing the protein solution frozenat −20° C. for about 2 months between the time of rabbit immunizationand affinity-purification of antiserum, indicating the long-termstability of purified His-ΔMTS-hCOQ5 protein.

Comparison on the Degree of Protein Aggregation and Protein Yield forHis-hCOQ5 Purified Under Denaturing Condition and His-ΔMTS-hCOQ5 ProteinPurified Under Native Condition Following Buffer Exchange with PBS

After protein was concentrated by using Vivaspin 6 UltrafiltrationDevice (GE) about 2 ml, protein concentration of concentrated proteinwas first determined by measuring absorbance at 280 nm. The proteinrecovery up to this step was calculated to be 10.2 mg and 25.3 mg forHis-hCOQ5 and His-ΔMTS-hCOQ5, respectively. A fraction of concentratedprotein with the same volume and same absorbance for two proteins wastaken for further buffer exchange for comparison. For each bufferexchange with PBS, 1 mL of concentrated protein was mixed gently with333 μL of PBS followed by centrifugation to about 1 ml. The same stepswere repeated for another four times. The amount of PBS was less here toensure gradual removal of urea for His-hCOQ5.

Right after four times of buffer exchange was completed, the His-hCOQ5protein solution purified under denaturing condition exhibited highturbidity, but the His-ΔMTS-hCOQ5 protein solution purified under nativecondition remained in clear form. The results of wavelength scan between200 nm to 800 nm for these two protein solutions with 10-fold dilutionin PBS was performed to demonstrate the presence of insoluble proteinaggregate, which could scatter light at all wavelengths, in His-hCOQ5protein solution, but not His-ΔMTS-hCOQ5 protein, could be observed(FIG. 3A). The marked turbidity and precipitation persisted in His-hCOQ5protein solution after the fifth buffer exchange was completed. Aftercentrifugation at a high speed (12000×g), there was a large pellet inthe tube with His-hCOQ5 protein, but not His-ΔMTS-hCOQ5. The differencebetween two protein solutions before centrifugation and aftercentrifugation could easily be visualized (FIG. 3B). The absorbance ofsupernatant at 280 nm for His-hCOQ5 solution and His-ΔMTS-hCOQ5 with10-fold dilution was 0.1207 and 0.6214, respectively. Therefore,starting from 2 L of bacterial culture, the yield of purified His-hCOQ5and His-ΔMTS-hCOQ5 protein recovered after 5 times of buffer exchangewas 1.5 mg and 17.6 mg, respectively. Therefore, it was much easier andcheaper to use soluble His-ΔMTS-hCOQ5 as the immunogen for production ofanti-hCOQ5 antibody and further purification of antibody by affinitycolumns.

Detection of Human COQ5 Protein in Human 143B Cells by Western Blot withthe Anti-COQ5 Antibody

The ability of this antibody to detect full-length and predicted matureform of endogenous human COQ5 protein was further examined by Westernblot analysis by using cell lysate, cytosolic fraction, andmitochondrial fraction from human 143B cell line. Results of Westernblot shown in FIG. 4 showed that both the full-length form (37.1 kD) andpredicted mature form (31.8 kD), which was the major form of endogenousCOQ5 protein in human cells, could be detected in cell lysate bypolyclonal antibody produced with the purified recombinant ΔMTS-hCOQ5protein. Full-length COQ5 protein (FL-COQ5) could be detected in bothcytosolic and mitochondrial fraction (Mito) and the levels were not verydifferent among total lysate, cytosol, and mitochondria. Mature form ofhuman COQ5 (ΔMTS-COQ5) could be detected in the mitochondria, but not inthe cytosol, and the level was enriched in the mitochondria comparedwith that of total lysate (FIG. 4).

To further prove that the two protein bands with molecular weight of37.1 kD and 31.8 kD detected by the affinity-purified antibody in FIG. 4was indeed full-length human COQ5 protein and mature COQ5 protein,respectively, the strategies of overexpression and knockdown of COQ5gene were employed.

As shown by FIG. 5A, in the experiment of transient overexpression ofCOQ5 gene by transient transfection of a plasmid expressing COQ5 genefor 72 hr, the transfection of vector alone (Vector) did not affect COQ5levels compared with that of non-transfected cells (NT). However, thesample transfected with COQ5 gene (COQ5) had markedly higher levels ofΔMTS-COQ5 (increased intensity of signal for the 31.8-kD band), but notthat of FL-COQ5 (the 37.1-kD band), in 143B cells, proving the identityof the 31.8-kD band. Next, we knocked down expression of COQ5 gene bytransiently transfection of small-interfering RNA (siRNA) for human COQ5gene into 143B cells. FIG. 5B shows that in the experiment for transientknockdown of COQ5 gene for 72 hr, transfection with negative-controlsiRNA did not affect levels of COQ5 protein compared with NT sample.However, transfection with COQ5 siRNA dramatically decrease levels ofboth ΔMTS-COQ5 (31.8-kD protein band) and FL-COQ5 (37.1-kD protein band)as detected by the antibody. The degree of decrease in full-length form(42.6% reduction) was less than that for mature form (89.9% reduction).Based on the results of experiments shown in FIG. 5, the antibodyproduced against the soluble recombinant human COQ5 protein wasdemonstrated to be specific for both precursor and mature form of humanCOQ5 protein.

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

What is claimed is:
 1. A producing and purifying method of solublerecombinant COQ5 protein expressed in non-denaturing conditions,comprising steps of: providing an E. coli host cell transformed with anexpression vector containing DNA fragments of encoding sequence forsoluble COQ5 protein; inducing the expressing of recombinant solubleCOQ5 protein in the E. coli host cell; lysing the bacterial cells by alysis buffer comprising lysozyme and DNase I; treating the cell lysatewith low concentration of ionic detergent; centrifugation for removinginsoluble proteins or debris and collecting supernatant containing therecombinant soluble COQ5 protein; and applying the supernatant to Ni-NTAbeads under native condition (without using urea) to obtain purifiedrecombinant soluble COQ5 protein.
 2. A soluble recombinant COQ5 proteinproduced by the method of claim 1, which is characterized by withoutappearance of aggregation or precipitation in protein solution afterbuffer replacement.
 3. The soluble recombinant COQ5 protein of claim 2,wherein the soluble COQ5 protein is a human COQ5 protein withoutmitochondrial targeting signal (MTS).
 4. The soluble recombinant COQ5protein of claim 2, wherein the said soluble COQ5 protein contains a Histag at N-terminus and comprises the amino acid sequence of SEQ ID NO. 2.5. The soluble recombinant COQ5 protein of claim 3, wherein the saidsoluble COQ5 protein contains a His tag at N-terminus and comprises theamino acid sequence of SEQ ID NO.
 2. 6. The soluble recombinant COQ5protein of claim 3, which is used to prepare anti-human COQ5 antibodies.7. The soluble recombinant COQ5 protein of claim 6, wherein theanti-human COQ5 antibody is applied to detect endogenous human COQ5protein in human cells or tissues.